Investigators in the early 50's to early 60's recognized that mucus in clinical samples could have adverse effects on clinical measurements carried out on the samples. In order to minimize the effect of mucous viscosity on clinical samples, specific methods and compositions have been developed to liquefy mucous matrices, such as sputum. Such extraction protocols include the use of DNAses (Lieberman, Amer. Rev. Resp. Disease, Vol. 97, pp. 662-672 (1968)) or thiol-containing reducing agents such as N-acetyl-L-cysteine (Webb, J. Thoracic & Cardiovascular Surg., Vol. 44, pp. 330-343 (1962)) or dithiothreitol (U.S. Pat. No. 3,502,779 (Mar. 24, 1970)). These reagents have been used alone or in combination (Lightowler & Lightowler, Arch. In Pharmacodyn., Vol. 189, pp. 53-58 (1971)).
There are a number of immunoassay or agglutination test kits for identifying viral particles, which have emerged on the market in recent years (QuickVue by Quidel, RSV Abbott TestPack by Abbott Laboratories, FluOIA by Biostar, Inc., and Directigen Flu and Directigen RSV by Becton Dickinson and Company). Most of the kits available on the market pre-treat clinical samples with a combination of a detergent and a reducing agent to break down the mucous matrix and to solubilize the antigens. Examples of antigen extraction using a combination of detergent and reducing agent are described in U.S. Pat. No. 6,248,513 to Horaud et al.; U.S. Pat. No. 6,207,445 to Crosby; U.S. Pat. No. 6,194,221 to Rehg et al.; U.S. Pat. No. 5,993,826 to Hansen et al.; U.S. Pat. No. 5,995,377 to Maul et al.; U.S. Pat. No. 5,869,272 to Bogart et al.; U.S. Pat. No. 5,415,994 to Imrich et al.; and U.S. Pat. No. 5,334,503 to Snyder et al., each of which are incorporated herein by reference. Alternatively, although rare, organic solvent extractions are also used in the art (U.S. Pat. No. 6,238,676 to Porcelli et al., which is incorporated herein by reference).
The detection of some bacterial strains via the detection of established protein markers can employ similar extraction procedures to those employed for viral particles. For example, to detect Legionella pneumophilia by immunochemical means, a swab containing a sputum sample is pretreated with extraction solution containing a Triton X-100 detergent and EDTA in phosphate-buffered saline (U.S. Pat. No. 5,415,994 (May 16, 1995)).
Nitrous acid has been used for the extraction of carbohydrate antigens from the cell wall of Streptococci and Enterococci (Petts, J. Clinical Microbiol., Vol. 33(4), pp. 1016-18 (1995); Facklam, Infections in Medicine, Vol. 14(11), pp. 891-898 (1997)). Additional examples include U.S. Pat. No. 5,494,801 to Bogart et al. and U.S. Pat. No. 5,536,646 to Sand et al., both of which are incorporated herein by reference. Nitrous acid extraction was only used for extracting cell wall-associated carbohydrate markers from these organisms. Nitrous acid extraction was never expected to be useful or successful in extracting other markers such as protein markers or markers from multiple types of microorganisms in a single sample, especially from a sample containing a complex biological matrix, such as a mucous excretion. On the contrary, it would have been expected that the use of nitrous acid would be detrimental to the assay of protein markers. Proteins undergo denitrification (loss of amine groups) when treated with nitrous acid. This decomposition pathway would be expected to lead to denaturation and/or precipitation. It may also change the antigenic properties of the protein (i.e., the antibodies may not be able to bind to the surface of the protein, since antibody binding characteristics change when the amines are removed). The denitrification, denaturation and/or precipitation would be expected to reduce the amount and accessibility of soluble markers, leavings the amount of markers insufficient for analysis. In addition, it is generally accepted in the art that extensive exposure to acidic conditions can adversely affect antigen solubility or the reactivity of the antibodies utilized by detection techniques (U.S. Pat. No. Re. 33,850).